We plan to continue our crosslinking studies of glycerinated muscle fibers to clarify the radial disposition of the crossbridges of thick filaments. We have examined the arrangement of the crossbridges under rigor conditions in previous studies. We now plan to extend this investigation to activated contraction fibers to determine whether both the S-1 subunits (myosin heads) as well as the S-2 links are away from the thick surface during the contractile cycle. We will continue our experiments on the number of myosin molecules in the native thick filaments using the Scanning Transmission Electron Microscope (STEM) in collaboration with Dr. Michael Beer. At the present time we are isolating the S-2 region of frog muscle myosin to investigate the thermal stability of this segment in a cold blooded animal. The helix-coil transition theory of muscle contraction implicates this segment in force-generation and it is expected that a knowledge of the stability of the frog S-2 will be helpful in comparing the force generation to be expected with that observed in the mechanical transient experiments of Huxley and Simmons on this muscle. The proteolytic sensitive region of myosin is believed to play a key role in the radial movement of the S-1 subunits as they go through their contractile cycle. We are currently attempting to isolate this segment of myosin and to study its physico-chemical properties.